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Keywords:

  • Cre recombinase;
  • transgenic mice;
  • hair cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We made a transgenic mouse that expresses Cre recombinase activity in inner ear hair cells by using a modified bacterial artificial chromosome containing Prestin. Cre recombinase activity was restricted to inner and outer hair cells, a subset of vestibular hair cells, spiral and vestibular ganglia in the inner ear, and a subset of cells in the testis, epididymis, and ear bone. This mouse will be useful for hair-cell–specific gene targeting. Developmental Dynamics 231:199–203, 2004. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The Cre-loxP recombination system derived from bacteriophage P1 is a powerful tool to modulate gene activity in specific cell types. It can be used to inactivate or activate a target gene in a controlled temporal and spatial manner (Sauer, 1998). Hair cells are mechanotransducers in the inner ear that are crucial for hearing and balance, but so far, no hair-cell–specific Cre-expressing lines have been reported (Hebert and McConnell, 2000; Cohen-Salmon et al., 2002; Zuo, 2002). The use of modified bacterial artificial chromosomes (BACs) has provided a powerful method by which a reporter gene can be expressed in the central nervous system and peripheral nervous system in a pattern that recapitulates the endogenous pattern of the gene of interest in the BAC (Zou et al., 1999; Gong et al., 2003). We aimed to drive Cre expression specifically in hair cells by using a BAC containing the Prestin gene, which encodes the motor protein of outer hair cells that is important for cochlear amplification (Zheng et al., 2000; Liberman et al., 2002; Maison et al., 2002).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We modified a BAC (427N18 of 129SV background) comprising approximately 150 kb of genomic DNA and containing the Prestin gene (∼50 kb) by inserting an internal ribosome entry site (IRES)/Cre cassette after its stop codon (Fig. 1A). We also inserted a (His)6-Flag tag immediately before the stop codon for biochemical use. BAC DNA was purified and injected (at 0.8 ng/μl) into the pronuclei of fertilized eggs from FVB/NJ mice. The transgenic founder was identified by using polymerase chain reaction (PCR) with primers derived from the BAC vector and Cre. Its identity was confirmed by Southern analysis and fluorescent in situ hybridization (FISH) analysis of interphase cells (Fig. 1B,C; Maison et al., 2002). The BAC transgene has a copy number of 1. All transgenic mice were fertile and behaviorally normal; they displayed approximately normal hearing thresholds (determined by their auditory brain stem responses to click stimuli), and their inner ears appeared morphologically normal (data not shown).

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Figure 1. Structure of the modified bacterial artificial chromosome (BAC) containing Prestin and characterization of the transgenic mice. A: Site-directed mutagenesis of a BAC containing the mouse Prestin gene. A (His)6-Flag tag was inserted before the stop codon in exon 20 of Prestin. This tag is followed by the stop codon and an IRES/Cre cassette. The IRES/Cre cassette contains an additional XbaI (X) site. Exon 3 contains the ATG initiation codon. B: Genomic Southern analysis of transgenic (Tg) and wild-type (Wt) mice by using an XbaI restriction digest and probes for Cre and exon 20. The intensity of the band representing the transgene (5.1 kb) is approximately half that of the band representing the endogenous Prestin gene segment (4.2 kb), a ratio that indicates the presence of one copy of the transgene. C: Fluorescent in situ hybridization analysis of tail cells, at interphase, from transgenic (Tg) and wild-type (Wt) mice by using Prestin BAC DNA as a probe. Prestin BAC DNA hybridized (green, arrows) to two endogenous Prestin loci on chromosomes 5 (in both Tg and Wt) and to a third transgenic locus (only in Tg). Similar intensities of the three spots are consistent with the presence of one copy of the BAC transgene. UTR, untranslated region; IRES, internal ribosome entry site.

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The transgenic FVB/NJ mice were subsequently mated with a reporter mouse line, ROSA26R (homozygous for lacZ), which has a mixed background of 129S4/SvJaeSor, C57Bl/6J, and FVB/NJ (Soriano, 1999; Kwon et al., 2001). ROSA26R mice express lacZ only when Cre recombinase is activated. We used three independent methods to verify the Cre recombinase-mediated lacZ expression among the offspring of this cross.

First, we looked for evidence of Cre recombinase activity in various tissues, including inner ear, brain, eye, heart, lung, liver, spleen, kidney, testis, and epididymis, by amplifying genomic DNA from these tissues with PCR primers flanking the two loxP sites in the lacZ locus of ROSA26R mice at postnatal days 0 (P0), 3, 9, 14, 30, and 60. Cre recombinase activity was first evident (as shown by the presence of PCR products) at P9 in the inner ear and at P14 in testis and epididymis but was absent from the other tissues examined (data not shown). This pattern of Cre recombinase activity is consistent with, albeit slightly delayed relative to, the reported pattern of Prestin expression (Zheng et al., 2000, 2003; Judice et al., 2002; Adler et al., 2003). Second, to visualize the expression pattern of β-galactosidase, an enzyme encoded by the ROSA26R locus, we used two methods: immunofluorescence with an antibody specific for β-galactosidase and histochemistry with X-gal, a substrate for β-galactosidase.

In offspring positive for Cre (i.e., Cre+), β-galactosidase was first detectable at P6 in most outer hair cells in the middle and basal turns, a few outer hair cells in the apical turns, some inner hair cells of the apical and basal turns, and a few inner hair cells in the middle turns of the cochlea. This pattern of outer hair cell β-galactosidase staining persisted at P60, whereas the number of inner hair cells in the middle turns that were positive for β-galactosidase gradually increased from P6 to P60 (Fig. 2A–D, data not shown). We observed both β-galactosidase immunofluorescence and histochemistry in the cytoplasm, including the perinuclear region, but not in the nuclei of hair cells (Fig. 2). These cytoplasmic compartments were likely lysosomes that had been previously reported in other β-galactosidase reporter mice (Oberdick et al., 1994) and are consistent with the finding that no nuclear localization signal exists in the lacZ gene at the ROSA26 locus (Soriano, 1999).

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Figure 2. β-Galactosidase immunofluorescence and X-gal staining in cryosections of various tissues from Cre+ and Cre− mice. A,C,E,G,I show β-galactosidase immunofluorescence at postnatal day 30; and B,D,F,H show X-gal staining at postnatal day 9. J shows X-gal staining at postnatal day 30. A,B: Middle turns of the cochlea from Cre− mice. C,D: Middle turns of the cochlea from Cre+. E–J: Various tissues from Cre+ mice. E,F: Spiral ganglia. G,H: Vestibular crista ampullaris. I,J: Epididymis. Scale bars = 20 μm in A–J.

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Furthermore, we detected β-galactosidase in a subset (∼10–20%) of spiral ganglia, vestibular hair cells, and vestibular ganglia at P6 to P60 (Fig. 2E–H). This pattern of Cre recombinase activity in a subset of vestibular hair cells is consistent with that of Prestin expression (Adler et al., 2003).

Whereas other tissues were negative, we found β-galactosidase–positive cells in testis and epididymis (Fig. 2I,J), again consistent with localization of Prestin mRNA (Judice et al., 2002; Zheng et al., 2003) and our genomic PCR results described above. Moreover, a subset of cells (likely osteocytes) in the bone surrounding the inner ear were positive starting at P6, and the intensity of staining in these cells appeared correlated with that in the hair cells (data not shown). Occasionally we observed a small number of cells also positive for β-galactosidase scattered in cochlear sections (data not shown); the staining was inconsistent among different slides, ages, and methods.

The pattern of Cre recombinase activity we have observed in this transgenic line is largely consistent with the known expression pattern of Prestin. This similarity provides evidence that the modified BAC transgene may contain the regulatory elements necessary to drive Prestin and Cre expression. However, the presence of Cre recombinase activity in inner hair cells, in a subset of spiral ganglia, and in ear bone cells was unknown previously. Because the Cre recombinase in our Prestin BAC transgene is extremely effective at inducing lacZ expression (i.e., a few Cre molecules are sufficient) and because its activity provides a record of the history, rather than the current state, of Prestin expression, our results indicate that Prestin may be expressed at low levels or transiently during development in inner hair cells, a subset of spiral and vestibular ganglia, and ear bone cells. It has been also reported that Prestin is expressed in brain at high levels (Zheng et al., 2003). However, in our Cre transgenic line, we did not detect any Cre activity by any of the methods we used (Fig. 3). This result suggests that our Prestin BAC transgene is either rearranged in the mouse genome or missing several regulatory elements necessary for the proper expression of Prestin in brain. Of interest, Cre recombinase-mediated lacZ expression in outer hair cells occurred later (starting between P3 and P6) than that of endogenous Prestin (starting at P2), a finding consistent with an expected delay between Cre transcription and Cre recombinase-activated lacZ expression.

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Figure 3. Absence of Cre activity in brain. A: In upper panel, genomic DNA was amplified with primers flanking the two LoxP sites of ROSA26 locus. Lane 1: brain, Cre+; lane 2: cochlea, Cre+; lane 3: liver, Cre+; lane 4: brain, Cre−. In the lower panel, the same genomic DNA samples were used for polymerase chain reaction amplification with a control primer pair (5′-CTTCCATCTGTGGCATACAG; 5′-CACAGCATCACCAGGCTGG) from the mouse Rp1 locus. B: β-Galactosidase immunostaining was performed in cryosections of cerebell from Cre+ and Cre− mice using diaminobenzidine staining and hematoxylin counterstaining. For positive control, another independent Cre-expressing line, which shows brain expression pattern, is used (unpublished data). An arrow indicates the positive staining of a Purkinje cell. Scale bars = 20 μm in B.

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In this transgene, a (His)6-Flag tag was inserted before the stop codon in exon 20 of Prestin; however, we failed to detect epitope-tagged Prestin or Cre by Western blot and immunofluorescence analyses by using either His tag or Cre antibodies (Novagen, data not shown). This result suggests that the expression level of our transgene is very low. The low level of Cre expression could be the key for the restricted pattern of Cre activity in cochlea. Furthermore, the low level of epitope-tagged Prestin may warrant that crossing with our Cre transgenic line for hair-cell–specific gene manipulation will not exert any significant impact in physiological analysis.

It has been difficult to obtain Cre recombinase activity specifically in hair cells that are separable from nearby supporting cells (Hebert and McConnell, 2000; Cohen-Salmon et al., 2002; Zuo, 2002). The Cre transgenic line here offers a valuable resource for the manipulation of genes in hair cells of the organ of Corti; such manipulation can be achieved by simply crossing our Cre transgenic line with mice containing a gene of interest engineered with two loxP sites. Because inactivation of genes specifically in hair cells by using our Cre transgenic line will start around P6 and, therefore, may not disrupt the early development of hair cells, our Cre transgenic line will allow study of many genes that have important roles in the maturation of hair cells.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Modification of the Bacterial Artificial Chromosome Containing Prestin

We screened the mouse BAC library (Catalogue no. 96050, Research Genetics, Huntsville, AL) in 129SV background with a cDNA clone encoding the Prestin gene. BAC clone, 427N18 was isolated and further characterized by Southern and PCR analysis. An IRES and a gene encoding Cre recombinase were inserted after the stop codon in exon 20 of Prestin. We inserted only one copy of the sequence encoding the (His)6-Flag tag at the 3′end of the coding region before the stop codon in exon 20. We modified BACs as previously described (Yang et al., 1997; Zuo et al., 1999).

Generation of Prestin–Cre Transgenic Mice

All modified BACs were purified by using a BAC Maxi Kit (Sigma) followed by filtration through a 0.45-μm filter and a Sepharose CL4B column. Then, it was equilibrated with Spermine and Spermidine for 48 hr and then injected into the FVB/NJ fertilized oocytes at a concentration of 0.8 ng/μl. The transgenic founder was identified by using PCR with primers derived from the BAC vector (5′-TAACTATGCGGCATCAGAGC-3′ and 5′-GCCTGCAGGTCGACTCTAGAG-3′) and Cre (5′-TGCAACGAGTGATGAGGTTC-3′ and 5′-ACGAACCTGGTCGAAATCAG-3′). PCR was performed using the following conditions: 95°C for 5 min, 30 cycles of 94°C 30 sec, 55°C 30 sec, and 72°C 30 sec and 72°C 5 min. Southern and FISH analysis was performed as previously described (Zuo et al., 1999).

Detection of Recombined Alleles by PCR

Various tissue DNA, including cochlea, brain, heart, liver, spleen, lung, testis was extracted and PCR was performed by using primers flanking the two loxP sites in the lacZ locus of ROSA26R mice (5′-GTCCAGGGTTTCCTTGATGA-3′ and 5′-GTGCTGCAAGGCGATTAAGT-3′). PCR was performed using the following conditions: 95°C for 5 min, 30 cycles of 94°C 30 sec, 55°C 30 sec, and 72°C 30 sec and 72°C 5 min. The PCR product is 317 base pairs (Fig. 3).

β-Galactosidase Immunostaining and X-Gal Staining

Animals at P3, 6, 9, 14, 21, 30, 60, and 150 were perfused with cold 1× phosphate-buffered saline (PBS) and then 2% paraformaldehyde. Inner ears and other tissues were dissected and post-fixed overnight in 2% paraformaldehyde. Cochleae from P14 and older mice were decalcified with 120 mM ethylenediaminetetraacetic acid (48–72 hr). Tissues and decalcified cochleae were incubated in 30% sucrose overnight, embedded, and cryosectioned at 12 μm. Sections were stored in −20°C refrigerator until use. For β-galactosidase immunofluorescent analysis, sections were washed in PBS and blocked by 10% goat serum/PBS for 1 hr; primary antibody was diluted in 1:5.000 (ICN Biomedicals) and was applied to sections at 4°C overnight. Sections were then washed with PBS and incubated with secondary antibody in the dark with goat anti-rabbit Alexa488 (Molecular Probes, 1:200 dilution). Sections were washed and covered with Fluoromount-G (Southern Biotechnology). Differential interference contrast images and fluorescent images were taken on a Carl Zeiss confocal microscope and superimposed. Biotin-labeled secondary antibody from ABC reagent (Vector Laboratories, Burlingame, CA) was also used according to the manufacturer's specifications. The tissue sections were incubated in diaminobenzidine for appropriate time and counterstained with hematoxylin.

X-gal histochemistry was performed on cryosections at and before P14 without intensive decalcification. Sections were air-dried for 10 min and washed in PBS twice. Sections then were incubated in X-gal Staining Set (Roche) at 37°C overnight. Then sections were washed again in PBS and covered for direct viewing. In all experiments, mice lacking Cre transgene (i.e., Cre−) served as negative controls.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank X. Wu, J. Gao, J. Treadaway, and J. Swift for assistance; and S. Baker and C. Kown for providing the ROSA26R mouse line and help in lacZ analysis. J.Z. and B.F. were funded by NIH grants.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
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